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Research is presented describing a study of N,N,N-tris(p-(N-oxyl-N-tert-butylamino)phenyl)amine, -borane and -methyl. Molecular structure and X-ray crystal analysis of the monoclinic single crystal shows it does not have a zwitterionic quinonoid structure or C3 symmetry.

The article presents a study on the triplet carbenes dialkylcarbenes, arylcarbenes and diarylcarbenes. The study finds that only a spin-delocalizing substituent can be useful to stabilize triplet carbenes. Also, carbene generated from diazo precursor should also have the space to quench by accepting a reagent externally such that a secondary protector should be introduced after the diazo group is introduced. Moreover, the triplet carbenes were found to have numerous applications.

Abstract: Catalase was confined in a nanospace of about 7nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material), forming a catalase-FSM conjugate. That the enzyme was encapsulated in the pores of mesoporous materials was proven by the direct visualization technique. The catalase-FSM showed not only an activity similar to that of the native catalase in the decomposition of H2O2 but also much higher stability: specifically, the catalase immobilized in FSM still retained its original activity after being applied 50 times to the decomposition, whereas the native lost its activity after 20 times recycling. Furthermore, the catalase-FSM showed catalase activity even in an organic media in which the native catalase had almost no catalytic activity. [Copyright &y& Elsevier]

Abstract: Enzymes, especially those known as membrane proteins existing in plasma membranes, direct important and complicated reactions in living bodies. Thus, attempts have been made to extract such enzymes from living bodies, and immobilize and accumulate them on supports to effectively use their functions for catalysis [M. Hartmann, Chem. Mater. 17 (2005) 4577–4593]. However, enzymes extracted from living bodies tend to aggregate in the absence of detergents or at high concentrations, resulting in a loss of their activities [Y. Urabe, T. Shiomi, T. Itoh, A. Kawai, T. Tsunoda, F. Mizukami, K. Sakaguchi, ChemBioChem 8 (2007) 668–674]. We have, however, succeeded in assembling a highly durable membrane capable of high-density accumulation and providing a regular array of catalase by encapsulating it in mesoporous silica synthesized in the pores of an alumina membrane. The artificial biomembrane showed not only activity similar to that of the native catalase for the decomposition of H2O2 but also much higher stability; the catalase immobilized in the membrane still retained its original activity even after being employed 160 times in decomposing H2O2, whereas the native lost its activity after 40 cycles. [Copyright &y& Elsevier]

Abstract: Choline sensor is successfully prepared by using immobilized enzyme, i.e., choline oxidase (ChOx) within a hybrid mesoporous membrane with 12nm pore diameter (F127M). The measurement was based on the detection of hydrogen peroxide, which is the co-product of the enzymatic choline oxidation. The determination range and the response time are 5.0–800μM and approximately 2min, respectively. The sensor is very stable compared to the native enzyme sensor and 85% of the initial response was maintained even after storage for 80 days. These results indicate that ChOx is successfully immobilized and well stabilized, and at the same time, enzyme reaction proceeds efficiently. Such ability of hybrid mesoporous membrane F127M suggests great promise for effective immobilization of enzyme useful for electrochemical biosensors. [Copyright &y& Elsevier]

We examined 24 microsatellite markers to select those most suitable for individual identification and parentage tests of brown bears ( Ursus arctos) based on statistical parameters and experimental error using liver samples (38 issues) from nuisance bears killed in the Akan–Shiranuka Region, eastern Hokkaido, Japan from 1996 to 2006. We found seven microsatellite markers suitable for identifying individuals (G1A, G10B, G10L, UarMU5, UarMU23, UarMU50, and UarMU51; probability of identity Pid = 3.17 × 10−7; probability of identity of sibs Pid-sib = 2.23 × 10−3), 12 microsatellite markers suitable for parentage testing when one parent is known [G1A, G1D, G10B, G10L, G10P, UarMU5, UarMU23, UarMU50, UarMU51, UarMU59, UarMU61, and UarMU64; probability of excluding one parent (PE I = 0.9991)], and 15 microsatellite markers for parentage testing when neither parents are known [three markers added to the above: G10M, G10X, and UarMU9; probability of excluding both parents (PE II = 0.9869)]. [ABSTRACT FROM AUTHOR]

Abstract: Mesoporous silica materials have a potential application for enzyme immobilization, which increases the stability of enzymes. We report the development of a novel detection method based on biosensors comprising an immobilized enzyme in mesoporous silica materials (i.e., FSM8.0 or P123-M), an electrochemical mediator (i.e., quinone), and an electrochemical cell, using the enzyme formaldehyde dehydrogenase. These biosensors exhibit a rapid response and high sensitivity, and they can detect 1.2μM of formaldehyde in an aqueous solution (corresponding to sub-ppb atmospheric concentration of formaldehyde). Furthermore, the sensors exhibit high selectivity, reusability, and a remarkable storage stability (stable over 80 days), indicating that formaldehyde dehydrogenase retains its highly ordered structure in these mesoporous silica materials. These results indicate that mesoporous silica materials can provide favorable methods for enzyme immobilization on the electrode and they are useful for developing high-performance electrochemical biosensors. [Copyright &y& Elsevier]

Using a working perfused heart model, we investigated the hypothesis that alterations in the NO–cGMP pathway may exacerbate postischaemic mechanical dysfunction in the hypertrophied heart. Ischaemia for 25 min followed by reperfusion for 30 min produced marked cardiac mechanical dysfunction in both stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY). Exogenous treatment with S-nitroso- N-acetyl-dl-penicillamine (SNAP), a NO donor, had beneficial effects on the cardiac dysfunction induced by ischaemia–reperfusion (I/R) in the WKY heart, but the cardioprotective effect of SNAP was eliminated by guanylyl cyclase inhibitor. Cardiac cGMP levels were increased by SNAP or ischaemia in WKY. In contrast, in SHRSP hearts, SNAP could not alleviate the cardiac dysfunction caused by I/R. Pre-ischaemia, the cardiac cGMP level was significantly higher in SHRSP than in WKY; however, no significant difference was found after SNAP and ischaemia. The myocardial Ca2+-dependent NO synthase (NOS) activity increased at the end of ischaemia in WKY. Conversely, the Ca2+-independent NOS activity and protein levels were upregulated by I/R in the SHRSP myocardium. In the SHRSP hearts, non-selective NOS and selective Ca2+-independent NOS inhibitors or antioxidant treatment alleviated cardiac dysfunction caused by I/R. Moreover, mRNA expression and Western blotting analysis of cGMP-dependent protein kinase type I showed more deterioration of SHRSP hearts compared with WKY. These results suggest that: (1) the NO-dependent cardioprotective effect is depressed; and (2) overproduction of NO derived from Ca2+-independent NOS contributes to postischaemic heart injury in the hypertrophied heart of hypertensive status. [ABSTRACT FROM AUTHOR]

Two types of diphenyldiazomethanes having two trifluoromethyl and two bromine groups at the ortho positions, either in unsymmetrical or in symmetrical fashion, that is, (2,6-dibromo-4-phenylphenyl)-[4-phenyl-2,6-bis(trifluoromethyl)phenyl]diazomethane (U-1—N2) and bis(2-bromo-4-phenyl-6-trifluoromethylphenyl)diazomethane (S-1—N2), are prepared. Triplet diphenylcarbenes (U-³1 or S-³1) are generated from those precursors and are characterized by ESR, UV/vis spectroscopy at low temperature, as well as time-resolved UV/vis spectroscopy at room temperature. Those carbenes are shown to be at least 2 orders of magnitude less reactive than the most stable triplet diphenylcarbene thus far known, that is, bis(2,6-dibromo-4-phenylphenyl)carbene. It has been also shown that S-³1 is significantly more stable than U-³1 even though both have the same two kinds of substituents. It is suspected that the perpendicular alignment of the two most bulky groups is a more effective way to shield the carbenic center than the planar one. By this way, triplet substituted diphenylcarbene surviving nearly a day in solution at room temperature is realized for the first time. [ABSTRACT FROM AUTHOR]

To confirm whether high-spin species can be generated as a result of ferromagnetic interaction between the 3d spin of metal ions and the 2p spins of triplet carbene through the pyridyl group located remote from the carbene center, [2,6-dibromo-4-(3- and 4-pyridyl)phenyl](4-tert-butyl-2,6-dimethylphenyl)diazomethanes were prepared and the corresponding carbenes were generated either in the absence or presence of Cu(hfac)2. These were characterized by ESR and UV/vis in a matrix at low temperature, and by laser flash photolysis in solution at room temperature. These studies indicated that although both carbenes generated a fairly stable complex with copper ions, the 4-pyridyl isomer formed a high-spin species as a result of ferromagnetic interaction between the 3d spin of metal ions and the 2p spins of triplet carbene. Such an interaction in the corresponding 3-isomer is likely to be antiferromagnetic. This is further confirmed by magnetic measurements using a Superconducting Quantum Interference Device (SQUID). The results demonstrate that extension of this method will enable stable high-spin polycarbenes to be obtained. [ABSTRACT FROM AUTHOR]

Diphenyldiazomethanes (2a-N2) having phenyl groups at the ortho positions were prepared and the corresponding diphenylcarbenes (2a) photolytically generated from them were characterized not only by the traditional product analysis method but also by ESR and UV/vis spectroscopy in a rigid organic matrix at low temperature and in laser flash photolysis in solution at room temperature. Product analysis indicated that fluorenes 4a are formed almost exclusively. Fluorene is most likely produced by the attack of singlet carbene ¹2a on an ortho carbon of the phenyl substituent to generate isofluorene, followed by 1 ,5-H shift. Irradiation of 2a-N2 in a 2-methyltetrahydrofuran (2-MTHF) matrix at 77 K gave ESR signals ascribable to triplet carbenes (³2a). UV/vis spectra of ³2a were obtained by irradiating 2a-N2 under identical conditions. However, laser flash photolysis (LFP) of 2a-N2 in degassed benzene at room temperature showed transient absorption bands completely different from those observed in photolysis in the 2-MTHF matrix at 77 K. The transient band decayed in first-order with a rate constant of 7.6 x 104 s-1, which was not retarded by deuterium substitution of o-phenyl hydrogens. Essentially the same transient band was observed in LFP of fluorene 4a. The LFP of 2a-N2 in nondegassed benzene gave transient absorption bands ascribable to triplet carbene ³2a and the corresponding carbonyl oxide. The quenching rate constant of ³2a by oxygen and the lifetime of ³2a in the absence of oxygen were estimated by plotting the observed pseudo-first-order rate constant of the formation of the carbonyl oxide against the concentration of oxygen. They were 1.9 × 107 M-1s-1 and 16 μs, respectively. Similar studies with diphenyldiazomethanes having a series of substituents (4-Ph, 3,5-(CF3)2, and 2-F) on the ortho-phenyl rings gave essentially identical results, indicating that those substituents exhibit little effect on overall reaction pathway. From those studies, it is suggested that triplet carbenes ³2 are also trapped by the ortho-phenyl ring to give eventually fluorenes. [ABSTRACT FROM AUTHOR]

The brain cAMP regulating system and its downstream elements play a pivotal role in the therapeutic effects of antidepressants. We previously reported the increase in activities of phosphodiesterase 4, a major phosphodiesterase isozyme hydrolyzing cAMP, in the frontal cortex and hippocampus of learned helplessness rats, an animal model for depression. The present study was undertaken to examine the combination of effects of rolipram, a phosphodiesterase 4 inhibitor, with imipramine, a typical tricyclic antidepressant, on depressive behavior in learned helplessness rats. Concurrently, cAMP-response element (CRE)-binding activity and brain-derived neurotrophic factor (BDNF) levels related to the therapeutic effects of antidepressants were determined. Repeated administration of imipramine (1.25–10 mg/kg, i.p.) or rolipram (1.25 mg/kg, i.p.) reduced the number of escape failures in learned helplessness rats. Imipramine could not completely ameliorate the escape behavior to a level similar to that of non-stressed rats even at 10 mg/kg. However, repeated coadministration of rolipram with imipramine (1.25 and 2.5 mg/kg, respectively) almost completely eliminated the escape failures in learned helplessness rats. The reduction of CRE-binding activities and BDNF levels in the frontal cortex or hippocampus in learned helplessness rats were ameliorated by treatment with imipramine or rolipram alone. CRE-binding activities and/or BDNF levels of the frontal cortex and hippocampus were significantly increased by treatment with a combination of rolipram and imipramine compared to those in imipramine-treated rats. These results indicated that coadministration of phosphodiesterase type 4 inhibitors with antidepressants may be more effective for depression therapy and suggest that elevation of the cAMP signal transduction pathway is involved in the antidepressive effects. [Copyright &y& Elsevier]

[9-[10-(4-t-Butyl-2,6-dimethyl)phenyl]anthryl](4-bromo-2,6-dimethylphenyl)diazomethane (1-N2) was found to be stable enough to survive under Sonogashira and Suzuki coupling reaction conditions, and bis(diazo) compounds incorporated into the 1,4-positions of butadiyne (3-2N2) and the 2,5position of thiophene (4-2N2) were prepared. Irradiation of those bis(diazo) compounds generated bis(carbenes), which were characterized by ESR and UV-vis spectroscopic techniques in a matrix at low temperature, as well as time-resolved U-V-vis spectroscopy in solution at room temperature. These studies revealed that both of the bis(carbenes), 3 and 4, have a singlet quinoidal diradical ground state with a very small singlet-triplet energy gap of less than 1 kcal/mol. A remarkable increase in the lifetime of bis(carbenes) as opposed to that of monocarbene (1) was noted and was interpreted to indicate that bis(carbenes) are thermodynamically stabilized as a result of delocalization of unpaired electrons throughout a π-net framework. Despite the stability, both bis(carbenes) are readily trapped by molecular oxygen to afford bis(ketones) as main products. [ABSTRACT FROM AUTHOR]

The present study examined the activities of adenylyl cyclase (AC) and rolipram-sensitive phosphodiesterase (PDE4) on brain regions in learned helplessness rat in order to clarify the cyclic AMP (cAMP) regulation system in the depressive state. Rats exposed to inescapable footshocks once a day for 3 days exhibited a significant increase in escape failure on Day 1 (the day after the last inescapable shock day) and Day 7. The plasma corticosterone level in rats subjected to inescapable shocks was significantly higher than that of nonstressed control rats on Days 1 and 7.The PDE4 activity of the frontal cortex was significantly lower than that of nonstressed control rats on Day 1. However, on Day 7, the PDE4 and [3H]-rolipram binding activities were significantly increased in the frontal cortex and hippocampus of learned helplessness rats compared with those of nonstressed control rats. Forskolin-stimulated AC activity was significantly decreased in the frontal cortex, hippocampus and striatum of learned helplessness rats on Day 1, but not on Day 7. Thus, a decrease in both AC and PDE4 activities was noted in the acute depressive state. In contrast, increase of PDE4 activity was noted in the delayed depressive phase, although no change of AC activity was observed. Gel shift assays also showed the decrease of cAMP-response element (CRE)-binding activity relating to cAMP activity in the frontal cortex and hippocampus of learned helplessness rats on Days 1 and 7. These findings indicated a delayed increase in PDE4 activity leading to hypofunction of the cAMP-dependent signal transduction system in the frontal cortex and hippocampus of learned helplessness rats, suggesting that up-regulation of the cAMP-degradation system by PDE4 may play a pivotal role in pathological states of chronic depression. [Copyright &y& Elsevier]